Hyaluronan synthases and methods of making and using same

Abstract

A functionally active hyaluronan synthase having at least one modified amino acid residue therein as compared to a corresponding functionally active native hyaluronan synthase such that the functionally active hyaluronan synthase has an altered enzymatic activity as compared to the corresponding functionally active native hyaluronan synthase is disclosed. Methods of producing hyaluronic acid utilizing a recombinant host cell having an expression construct encoding the functionally active hyaluronan synthase with altered enzymatic activity are also disclosed.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims benefit under 35 U.S.C. 119(e) of U.S. Ser. No. 60 / 336,105, filed Dec. 3, 2001, entitled “NOVEL KINETIC PROPERTIES OF HYALURONIC SYNTHASES”, the contents of which are hereby expressly incorporated herein by reference.[0002]This application is also a continuation-in-part of U.S. Ser. No. 10 / 011,771, filed Dec. 11, 2001, entitled “HYALURONAN SYNTHASE GENE AND USES THEREOF”; which is a continuation of U.S. Ser. No. 09 / 469,200, filed Dec. 21, 1999, entitled “HYALURONAN SYNTHASE GENE AND USES THEREOF”, now U.S. Pat. No. 6,833,264, issued Dec. 21, 2004; which is a continuation of U.S. Ser. No. 09 / 178,851, filed Oct. 26, 1998 now abandoned, entitled “HYALURONAN SYNTHASE GENE AND USES THEREOF,” now abandoned; the contents of each of which are hereby expressly incorporated herein by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT[0003]This application was supported in part by a grant from the Natio...

AI Technical Summary

Benefits of technology

[0053]FIG. 2. Effect of NEM concentration and incubation time on the activity of seHAS and spHAS. Panel A: E. coli membranes containing recombinant seHAS or spHAS were incubated at 4° C. for 1 h with Phosphate Buffered Saline (PBS) alone (minus N-ethylmaleimide (NEM) control) or PBS containing different concentrations of NEM. The unreacted NEM was quenched by addition of dithioerythritol (DTE) to a final concentration of 1–6 mM and the samples were assayed for HAS activity as described hereinafter. Panel B: The effect of incubation time on seHAS and spHAS activity was assessed by incubating the membranes with 5 mM NEM at 4° C. for the indicated times. Aliquots were removed into assay buffer containing 5 mM DTE, and HAS activities were determined. HAS activity in control untreated membranes was stable for 1 h at 4° C. The inhibition of HAS activity is expressed as percent relative to the controls.

[0054]FIG. 3. Effect of NEM or sodium arsenite treatment on the utilization of UDP-GIcUA and UDP-GIcNAc by wild-type seHAS. E. coli membranes containing seHAS protein were incubated at 4° C. for 1 h in PBS containing 5 mM NEM or 10 mM Sodium Arsenite (SodArs), and the control membranes were incubated with PBS alone. Michaelis-Menten constants (Km) were calculated from the activities of seHAS at varying concentrations of UDP-GIcUA or UDP-GIcNAc.

Problems solved by technology

The incidence of streptococcal infections is a major health and economic problem worldwide, particularly in developing countries.

The extrusion of the growing chain into the extracellular space also allows for unconstrained polymer growth, thereby achieving the exceptionally large size of HA, whereas confinement of synthesis within a Golgi or post-Golgi compartment limits the overall amount or length of the polymers formed.

High concentrations of HA within a confined lumen may also create a high viscosity environment that might be deleterious for other organelle functions.

Although the streptococcal and murine oligodendroglioma enzymes were successfully detergent-solubilized and studied, efforts to purify an active HAS for further study or molecular cloning remained unsuccessful for decades.

This led to a report claiming that the Group C streptococcal HAS had been cloned, which was unfortunately erroneous.

Despite these efforts, progress in understanding the regulation and mechanisms of HA synthesis was essentially stalled, since there were no molecular probes for HAS mRNA or HAS protein.

Unfortunately, several studies have employed antibodies to this uncharacterized 52-kDa streptococcal protein to investigate what was believed to be eukaryotic HAS.

It is generally felt that isolation of HA from rooster combs is laborious and difficult, since one starts with HA in a less pure state.

Unfortunately, very high molecular weight HA, such as that ranging up to 107, has been difficult to obtain by currently available isolation procedures.

However, to date the involvement of one or more of these conserved Cys residues in enzyme activity or disulfide bond formation has not been determined.

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